Triplex in-situ hybridization

ABSTRACT

Disclosed are methods for detecting in situ the presence of a target sequence in a substantially double-stranded nucleic acid segment, which comprises: a) contacting in situ under conditions suitable for hybridization a substantially double-stranded nucleic acid segment with a detectable third strand, said third strand being capable of hybridizing to at least a portion of the target sequence to form a triple-stranded structure, if said target sequence is present; and b) detecting whether hybridization between the third strand and the target sequence has occured.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a 371 of PCT/US98/23765 filed Nov. 10, 1998, whichclaims benefit of U.S. Ser. No. 60/064,997 filed Nov. 10, 1997.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The present work was supported by grants from the National Institutes ofHealth (GM42936) and the Department of Energy (DE-FG02-96ER62202).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods for identifying and classifyingnucleic acids by means of in situ hybridization of a third-strand probeto a duplex DNA target.

2. Description of Related Art

A list of the references referred to by number herein is found at theend of the Detailed Description of the Invention herein.

Oligodeoxy- and ribo-nucleotide ‘third strands’ bind in sequence- andpolarity-specific alignment within the major groove of cognatepurine-rich•pyrimidine-rich nucleic acid duplex target sequences (1). Athird-strand binding code has been elucidated (2) that encompassestriplexes of various structural motifs, of which the best characterizedis the pyrlmidine-parallel (Y:R•Y) with T:A•T and C⁺:G•C triplets. Thefidelity of third-strand binding to cognate targets of equal and lesserlength, targets contained within large DNA molecules, and targets withinverted base pairs, i.e., R•Y→Y•R, is well documented (3). Optimalsolution conditions for third-strand binding, i.e., pH, counterionicstrength, and temperature, can be manipulated to induce third-stranddissociation or prevent association. While certain triplet mismatchesare moderately tolerated within the Y:R•Y motif (4), e.g., T:C•G andG:T•A, others are especially destabilizing. Such destabilizationprovides temperature as a variable to selectively favor, under in vitroand demonstrated in situ conditions, desired triplexes over less perfectones, as required in the large non-specific duplex DNA background of thehuman genome, while maintaining essentially constant pH and ionicstrength.

The use of third strands for sequence-specific recognition of humangenomic DNA has been exploited for the development of potentialanti-gene therapeutic agents (5) and artificial endonucleases (6). Byvirtue of the present invention, this approach is extended to in situhybridization for the detection and analysis of nucleic acids.

SUMMARY OF THE INVENTION

The present invention provides a method for detecting in situ thepresence of a target sequence in a substantially double-stranded nucleicacid segment, which comprises:

a) contacting in situ under conditions suitable for hybridization asubstantially double-stranded nucleic acid segment with a detectablethird strand, said third strand being capable of hybridizing to at leasta portion of the target sequence to form a triple-stranded structure, ifsaid target sequence is present in the nucleic acid segment; and

b) detecting whether hybridization between the third strand and thetarget sequence has occurred.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts the organization of chromosome 17 ∝- satellite DNA. Atandem array of the chr.17 2.7 kb EcoRI (E) higher-order repeat, singlehigher-order repeat, and the sixteen 171 bp monomer units that compriseit. Gray rectangles represent the unique 16 bp third-strand targetsequence within each monomer sixteen.

FIG. 1B depicts the 16-nt third strand [SEQ ID NO.2] and its 16 bp∝-satellite target [SEQ ID NO.76] and [SEQ ID NO.77]. Third strand T andm⁵C⁺ residues are bound via Hoogsteen-hydrogen bonds (•) to the purineresidues of their Watson-Crick (|) target [SEQ ID NO.76]. The boxedsequence shows the upstream ∝-satellite DNA T residues capable offorming photoadducts with the psoralen(4′-hydroxymethyl-4,5′,8-trimethylpsoralen, HMT) moiety of thedual-modified third strand. Ps=psoralen; Bi=biotin.

FIG. 2A is an autoradiograph showing the specificity of third-strandbinding to supercoiled (sc) p17H8. Gel purified ³²P-end-labeled5′-Tm⁵C-3′ (8×10⁶ cpm/pmol) and 0.25 μg of 2×CsCl p17H8 or pMJ1 weremixed at 1:1 oligomer:plasmid molar ratio in 1×triplexcocktail±deionized formamide (0.25 or 2.5%) and yeast RNA (1 ng) at pH6.0 and 23° C. for ˜20 hrs to attain equilibrium. 25 μl rxn samples wereelectrophoresed on a 0.8% SeaKem Gold (FMC) agarose gel overnight at 23°C. and 1.3 V/cm with buffer recirculation. Pre-flashed film (DupontReflection) was exposed to the dried gel at 4° C. for 15 min. Controllanes 1 and 8 contain ³²P-third strand alone, lanes 2-4 ³²P-third strandand p17H8, and lanes 5-7 ³²P-third strand and pMJ1 (lacking target).

FIG. 2B is an autoradiograph showing rate-dependent third strand bindingto sc p17H8 in the presence of ˜2,400 unsorted human metaphasechromosomes (1-22, XY). Gel purified ³²P-end-labeled 5′-Tm⁵C-3′ (0.5×10⁶cpm/pmol) and 0.25 μg of 2×CsCl p17H8 were present at a 2:1oligomer:plasmid molar ratio. 25 μl reactions were carried out in1×triplex cocktail at pH 6.0 and 23° C. After 1.0, 2.5, and 5.0 hrincubations, the reactions were centrifuged at 3,000 rpm and 4° C. for30 minutes to pellet the chromosomes. The supernatants wereelectrophoresed as described above, and film exposure was for 7 hr. Lane1 contains ³²P-third strand and p17H8, but no chromiosomes. Lanes 2A,3A, and 4A represent reactions in which chromosomes, ³²P-third strand,and p17H8 were added together, while for lanes 2B, 3B and 4B,chromosomes and ³²P-third strand were preincubated together for 1 hrprior to adding p17H8.

FIG. 3 is a standard non-denatured metaphase spread and interphasenucleus prepared for TISH, but “hybridized” with a D17Z1-specific dsprobe using a conventional FISH protocol (Oncor, Inc.). Non-denaturedspreads and nuclei were hybridized at 37° C. for 1 hr with 5 ng of adenatured 200 bp biotin-11dUTP nick-translated probe (Oncor, Inc.). Nofluorescent signals are visible after FITC-avidin-based detection. Thechromosomes are stained with PI.

FIGS. 4A and 4B are TISH images of a non-denatured (A) metaphase spreadand (B) interphase nuclei hybridized wit the 16-nt chr. 17∝-satellite-specific third strand 5′Pso-Tm⁵C-Bio-3′. FITC-avidin-baseddetection clearly identifies two centromere-specific fluorescent spotson anatomically homologous metaphase chromosomes that are the expectedsize for chr.17. Each interphase nucleus shows two fluorescent spots.

FIGS. 5A-5C are TISH images of (A) a non-denatured mouse×human chr. 17metaphase spread and (B) nuclei hybridized with the chr. 17∝-satellite-specific third strand 5′-Pso-Tm⁵C-Bio-3′. FITC-avidin-baseddetection is specific for the single human chr.17s. FIG. 5C is a TISHimage showing a competition assay that demonstrates specificity ofthird-strand binding to chr.17 ∝-satellite in human metaphase spreads. A25:1 molar ratio of 5′-Tm⁵C-3′ to 5′-Pso-Tm⁵C-Bio 3′ effectivelycompetes out the dual modified third-strand. The white bar at the bottomleft of each micrograph represents 10 microns.

FIGS. 6A-6D are TISH and FISH images of metaphase spreads and interphasenuclei from two individuals differing in their TISH-based chr.17fluorescent signal patterns. The two haplotypes for third-strandbinding, +17 and −17, are shown in two combinations, (A) +17/+17 and (C)+17/−17. FIGS. 6(B) and 6(D) are their corresponding +17/+17 FISHimages.

DETAILED DESCRIPTION OF THE INVENTION

Third Strand

The third strand is a synthetic or naturally occurring oligonucleotidecapable of binding with specificity to a predetermined target region ofa double-stranded nucleic acid molecule to form a triple-strandedstructure. The third strand may bind solely to one strand of the nativenucleic acid molecule, or may bind to both strands at different pointsalong its length.

Preferably, the oligonucleotide probe is a single-stranded DNA moleculebetween about 7 and about 50, most preferably between about 10 and about23 nucleotides in length. Its base composition can be homopurine,homopyrimidine, or a mixture of purines and pyrimidines. Thethird-strand binding code and preferred conditions under which atriple-stranded helix will form are well known to those skilled in theart (U.S. Pat. No. 5,422,251; Beal and Dervan, Science 251: 1360 (1991);Beal and Dervan, Nucleic Acids Res., 20:2773 (1992); Broitman andFresco, Proc. Natl. Acad. Sci. USA, 84:5120 (1987); Fossella, et al.,Nuc. Acids Res. 21:4511 (1993); Letai, et al., Biochemistry 27:9108(1988); Sun, et al., Proc. Natl. Acad. Sci. USA 86:9198 (1989)).Briefly, adenosine, uridine, thymidine and inosine in the third strandwill bind to adenosine in the duplex, and guanosine, cytidine andinosine in the third strand will bind to guanosine in the duplex. Thethird strand need not be perfectly complementary (in the binding codesense, not in the Watson-Crick sense) to the duplex, but may besubstantially complementary. In general, by substantially complementaryis meant that one mismatch is tolerable in about every 10 base pairs.

The third strand may have a natural phosphodiester backbone or may becomprised of other backbone chemical groups or mixtures of chemicalgroups which do not prevent the triple-stranded helix from forming.These alternative chemical groups include phosphorothioates,methylphosphonates, peptide nucleic acids (PNAs), and others known tothose skilled in the art. Preferably, the third-strand backbone isphosphodiester.

The third strand may also comprise one or more modified sugars, whichwould be known to those skilled in the art. An example of such a sugarincludes α-enantiomers.

The third strand may also incorporate one or more synthetic bases ifsuch is necessary or desirable to improve third-strand binding. Examplesof synthetic base design and the bases so designed are found in theco-pending U.S. application Ser. No. 08/473,888 of Fresco, et al.entitled “Residues for Binding Third Strands to Complementary NucleicAcid Duplexes of any Base-Pair Sequence”, filed Jun. 7, 1995, andpublished as WO/9641009, the contents of which are incorporated hereinby reference.

If it is necessary to protect the third strand from nucleases residentin the target cells, the third strand may be modified with one or moreprotective groups. In a preferred embodiment, the 3′ and 5′ ends may becapped with a number of chemical groups known to one of ordinary skill,such as alkyl amines, acridine, cholesterol, etc. In another embodiment,the third strand may be protected from exonucleases by circularization.

Label

The third strand should include a reporter group which has a physical orchemical characteristic which can be measured or detected by appropriatedetector systems or procedures, which will allow detection of triplexformation. Detectability can be provided by such characteristics ascolor change, luminescence, fluorescence or radioactivity, or it can beprovided by the ability of the reporter group to serve as a ligandrecognition site. Such characteristics can be measured or detected, forexample, by the use of conventional calorimetric, spectrophotometric,fluorometric or radioactivity sensing instrumentation, or by visualinspection, e.g., microscopic observation.

The interactions which can be usefully initiated by the reporter groupinclude appropriately specific and selective interactions productive ofgroups or complexes which are themselves detectable, for example, bycolorimetric, spectrophotometric, fluorometric or radioactive detectionprocedures. Such interactions can take the form of protein-ligand,enzyme-substrate, antibody-antigen, carbohydrate-lectin,protein-cofactor, protein-effector, nucleic acid-nucleic acid, andnucleic acid-ligand interactions. Examples of such ligand-ligandinteractions include fluorescein-antifluorescein antibody,dinitrophenyl-dinitrophenyl antibody and biotin-avidin. Either one ofeach of such ligand pairs may serve as a ligand recognition typereporter group. Preferred reporter groups of the present inventioninclude, for example, biotin, fluorescein, digoxigenin, phenyloxazolone,tetramethyl rhodamine, Texas Red, and BODIPY (contains the4,4-difluoro-4-bora-3a,4a-diaza-s-indacene unit).

General methods for joining the reporter group to the third strand arewell known to those skilled in the art. Examples of those methods may befound, for example, in U.S. Pat. Nos. 4,711,955 and 5,684,142.

Target

The substantially double-stranded nucleic acid segment may comprise anycombination of naturally occurring nucleic acid types, such as two DNAstrands, two RNA strands, or a DNA-RNA hybrid. By “substantiallydouble-stranded” is meant that the two strands which make up the nucleicacid segment need not be hybridized to each other along their entirelength. It is preferred that the target sequence to which the thirdstrand binds is completely double-stranded, but a degree of non-bindingbetween the two strands of the target sequence is tolerable, so long asit does not exceed about 1 mismatch in 10 base pairs.

The target sequence may be any sequence for which detection is desired,and may be anywhere along the length of DNA or RNA found in a cell,including α-satellite regions, other satellite regions, non-satelliteheterochromatin, or euchromatin regions. The target sequence may bewithin the chromosomal region of a eukaryotic species, particularlyorganisms of commercial significance in agriculture, or thedouble-stranded stage of a virus.

The in situ hybridization may take place in environments known to thoseskilled in the art, and are in most respects similar to those used forconventional in situ hybridization on denatured nucleic acids, althoughaccording to the present invention the target sequence is not denatured.The present hybridization may be performed on metaphase spreads andinterphase nuclei, the preparation of which would be readily apparent tothose skilled in the art. The major difference from conventional in situhybridization is that in performance of the present invention, thehybridization conditions (e.g., temperature, pH,. salt concentration,etc.) are non-denaturing and should be such that, as described above,the target nucleic acid segment is substantially double-stranded.

It is preferred that the target sequence contain a purine-rich segmenton one strand and a complementary pyrimidine-rich segment on the otherstrand. A stretch of at least ten consecutive purine bases isparticularly preferred.

Binding Enhancement

The oligonucleotide may be modified to include a moiety which willenhance the binding of the third strand to the target sequence. Such amoiety may be positioned anywhere along the length of the third strand.Suitable moieties include well known DNA-binding, cross-linking orintercalating agents such as psoralen, acridine, coralyne, etc. Thebinding moiety is often incorporated into the oligonucleotide during itssynthesis. For example, commercially available compounds such aspsoralen C2 phosphoroamidite (Glen Research, Sterling Va.) are insertedinto a specific location within an oligonucleotide sequence inaccordance with the methods of Takasugi, et al., Proc. Natl. Acad. SciUSA, 88:5602 (1991); Gia, et al., Biochemistry 31:11818 (1992);Giovannangeli, et al., Proc. Natl. Acad. Sci. USA, 89:8631 (1992), allof which are incorporated by reference herein. The binding moiety mayalso be attached to the oligonucleotide through a linker, such assulfo-m-maleimidonbenzoly-N-hydroxysuccinimide ester (sulfo-MBS, PierceChemical Company, Rockford Ill.) in accordance with the methods of Liu,et al., Biochem. 18:690 (1979) and Kitagawa and Ailawa, J. Biochem.79:233 (1976), both of which are incorporated by reference herein.

Utility

Third-strand in situ hybridization (TISH) complements and extendsdenaturant Watson-Crick-based FISH (7) technology to permit molecularcytogenetic and biochemical studies of non-denatured metaphase andinterphase fixed chromosomes and chromatin, though it is not restrictedto such uses. In Table 1 below, we have identified unique multicopyα-satellite third-strand target sequences in 22 of 24 human chromosomes,making centromere-specific chromosome identification by TISH applicableto virtually all human chromosomes. In the Table, third-strand targetsequences (Column 2) within the ∝-Satellite DNA of a particular humanchromosome type or chromosome group, e.g., 1, 15, 16 (Column 1) aregiven. Numbers after the decimal indicate that different third-strandtarget sequences are shared within a group or are unique to a singlechromosome. Group (1, 15, 16) has three target sequences. Chromosome 3contains four uniquely different target sequences.

TABLE 1 Chromosome Type or Group Target Sequence 1.1 AGTAAAGGAA AGAA[SEQ ID NO:3] 1, 15, 16.1 TATTTCCTTT TCTCGCTT [SEQ ID NO:4] 1, 15, 16.2GAATGAAAAG GAAAG [SEQ ID NO:5] 1, 15, 16.3 TTTCCTTTTC TCGCTTT [SEQ IDNO:6] 1, 5, 19.1 AAAGGTAGAA AAGGAAATA [SEQ ID NO:7] 1, 5, 19.2AAAGGCAGAA AAGGAAATA [SEQ ID NO:8] 1, 5, 19.3 CTTTTCTTTT TCATTC [SEQ IDNO:9] 2.1 CCTTTCTTTT GAGAGAGCAG [SEQ ID NO:10] 2, 20.1 GAAAAAGGAAATATCTTCCC [SEQ ID NO:11] CT 3.1 TTTACCCCTT TCTTTTC [SEQ ID NO:12] 3.2GATAGAAAAG GAAA [SEQ ID NO:13] 3.3 GGTAGAAAAG GAAA [SEQ ID NO:14] 3.4CTTTCCTTTA GAAAACAGCA [SEQ ID NO:15] GAG 5.1 GGAAAAkAG GATA [SEQ IDNO:16] 5.2 CTTTCTCCTT ACTT [SEQ ID NO:17] 6.1 GAAAAGGGAG GTTTCACTCT [SEQID NO:18] TT 6.2 CTTTCTCTAC CAAAAGAAAG [SEQ ID NO:19] G 7.1 GGTGAAAATGGAAAAGGAAA [SEQ ID NO:20] 7.2 GAGGCAAATG GAGAAAAAG [SEQ ID NO:21] 7.3TCCTTTCTTT TCATTC [SEQ ID NO:22] 7.4 ACAGAGGAAA AGGAAA [SEQ ID NO:23]7.5 ATGGAGGAAA AGGAAA [SEQ ID NO:24] 9.1 AGAGATGAAC CTTTCTTTTT [SEQ IDNO:25] 10.1 ACGGGGAGAA AGGAAATA [SEQ ID NO:26] 10.2 ATGGAGAGAA AGGAAATA[SEQ ID NO:27] 10.3 AGAGGGAGCA GAGGTGAAA [SEQ ID NO:28] 10.4 TTTCTCCTTTCTCTTCAT [SEQ ID NO:29] 10.5 TTCCACCTTT CTTTTC [SEQ ID NO:30] 10.6CCTTTCTTGA GAGAGAGCAG [SEQ ID NO:31] A 10.7 TTTCACCTTT CTCTTC [SEQ IDNO:32] 10.8 CCTTCCTTTA GAGAGAGCAG [SEQ ID NO:33] A 10, 12.1 AAAGGTAGAAAAGGAAACA [SEQ ID NO:34] 10, 12.2 GAAGAGAAAG GTGAAA [SEQ ID NO:35] 10,12.3 CCTTTCTTTT GATGGAGGAG [SEQ ID NO:36] 10, 12.4 CTCTCTCTAA AGAAAGG[SEQ ID NO:37] 10, 12.5 AAAGGTAGAA AAGGAAATA [SEQ ID NO:38] 12.1CCTTTCTTTT GATGAAGGAG [SEQ ID NO:39] 12.2 GGAAACGGGA TTTCTTCCT [SEQ IDNO:40] 12.3 CCTTTCTTTT GATGAAGGAG [SEQ ID NO:41] 13, 21.1 GGTGAAAAAGGGAA [SEQ ID NO:42] 13, 21.2 GAAAAAGGGA ATGTCTTCCC [SEQ ID NO:43] 13,21.3 AGAGTGGAAC CTCTCTCTTT [SEQ ID NO:44] T 14, 22.1 GGTGAGAAAG GAAA[SEQ ID NO:45] 14, 22.2 AGAGGTGGAT CTTTCTTTT [SEQ ID NO:46] 14, 22.3AAAGGGAATA TCTTCCCCT [SEQ ID NO.47] 14, 22.4 GGTGAAAAGG GAAA [SEQ IDNO:48] 14, 22.5 GAAAAGGGAA ATATCTTCTC [SEQ ID NO:49] 14, 22.6 AACAGAGAAGAACCTTCCTT [SEQ ID NO:50] TT 16.1 TTTCACCTTT CTTTTC [SEQ ID NO:51] 17.1AGAAAGAAGA CAGAAG [SEQ ID NO:52] 17.2 AAAAAGAAGA CAGAAG [SEQ ID NO:53]17.3 TTTTTTTTCC TCTCT [SEQ ID NO:54] 17.4 CTTTCCTTTC GAGAGAAG [SEQ IDNO:55] 17.5 ITTTCCTTTC GAGAGAGAAG [SEQ ID NO:56] 17.6 GGAAAAGGAATTATCTTTCC [SEQ ID NO:57] C 18.1 AAGGTGAAAA AGGAAA [SEQ ID NO:58] 18.2ATGGAGAGAA AGGAAA [SEQ ID NO:59] 18.3 AAGGTGAAAA AGAAA [SEQ ID NO:60]18.4 AAAGGAGTAG AACCTTTCTT [SEQ ID NO:61] TTC 20.1 GGTGAAAAAG GAGA [SEQID NO:62] 20.2 GGTGAAAAAG GAAA [SEQ ID NO:63] 20.3 GAAAAAGGAA ATATCTTCCC[SEQ ID NO:64] 22.1 GGTGGAGAAG GAAA [SEQ ID NO:65] 22.2 GGAAAAGAATTATCTTCTC [SEQ ID NO:66] 22.3 GGTGGAAAAG GAAA [SEQ ID NO:67] 22.4AGTGGAAAAG GAAA [SEQ ID NO:68] 22.5 TTCCCTTTCA GAGAGCAG [SEQ ID NO:69]22.6 AAAGGAAATA TCTTCCCCT [SEQ ID NO:70] X.1 ACAGAAAGAC GAGAGAGAAG [SEQID NO:71] CA X.2 CCTTTTCCTT TATCTTC [SEQ ID NO:72] X.3 GGAAAAGGAAATATCTTCTC [SEQ ID NO:73] C Y.1 GGAAGATGGT GGAAAAGGAA [SEQ ID NO:74] AY.2 GGAAAAGGAA GTATCTTCCT [SEQ ID NO:75]

TISH is also applicable to other multicopy sequences, as well as tosingle copy sequence identification, if those sequences are amenable tothird-strand binding. In that event, they would have the same utility asFISH for diagnosing genetic disorders, screening for individuals at riskfor developing genetic-based diseases, and for diagnosing infectiousdiseases by detecting the presence of target sequences unique to apathogen. Complementary diagnostic roles for chromosome-specificα-satellite third-strand probes are as determinants of numericalchromosome abnormalities, i.e., aneuploidy (e.g., trisomy 21), inmetaphase and interphase cells of tumors, and of individuals withgenetic disorders. TISH-based aneuploid detection is particularlyadvantageous for those cell types having small nuclear volumes andextremely condensed chromatin, e.g., uncultured prenatal amniocyte fluidcells and spermatozoa, since interphase chromatin denaturation is notrequired, and third-strand penetration of such nuclei under solutionconditions may be more efficient. It is also noted that while FISH andG-banding are incompatible, the former requiring denatured chromosomalDNA and the latter non-denatured DNA, TISH shares that requirement withG-banding, so that those two techniques are applicable to the samemitotic chromosome preparations. In addition, the meiotic stability ofchromosome-specific α-satellite polymorphisms (8, 9) should, in caseswhere third-strand target sequences are heteromorphic, e.g., D17Z1,permit TISH to serve as a rapid assay for Mendelian segregationanalysis.

Third-strand hybridization of chromosome-specific α-satellite probes tointerphase chromatin within nuclei may provide a tool for studyingchromosome organization under aqueous conditions. Hybridization of suchprobes to native metaphase chromosomes within mitotic cells insuspension also makes possible third strand-based chromosome-specificunivariate flow sorting.

The use and novel features of the present invention will be furtherunderstood in view of the following non-limiting examples.

EXAMPLE 1 Selection of Chromosome 17 α-Satellite Target Sequence

GenBank, EMBL, and GB_New databases were searched for human o-satellitesequences using GCG software (10), and a Homo sapiens α-satellitedatabase was constructed from 336 sequence entries containing confirmednucleotide sequences. This database was then searched to identify samestrand and alternate strand (i.e., where the target sequence switchesfrom one duplex strand to the other) homopurine•homopyrimidine runs ofdefined length and specificity. Mismatches due to inverted base pairs(base pairs with a pyrimidine residue in the predominantlypurine-containing target strand) were limited to one per ten residues(2/20, 3/30, etc.); and the position (internal vs. terminal) andrelation (adjacent vs. separate) of two or more mismatches wasconsidered when purine-rich•pyrimidine-rich runs exceeded 19 residues.

Three same strand and three alternate strand target sequences of varyingspecificities were identified for chromosome 17. The nucleotide sequenceof the chromosome 17 2.7 kb α-satellite higher-order repeat inrecombinant clone p17H8 (11) was searched for matches to the sixpotential target sequences. Identical sequences were found inhigher-order repeat monomers 11 and 16 for one alternate and one samestrand target sequence, respectively. Similar sequences with mismatcheswere found for two others, one differing from the monomer 16 sequence bya single point mutation, and no matches were found for the remainingtwo. The unique 16 bp purine-rich•pyrimidine-rich chromosome 17α-satellite sequence located in monomer 16, and contained on p17H8, wasselected for in vitro and in situ third-strand targeting.

Molecular Organization of Chromosome 17 α-Satellite and Target

The predominant organization of α-satellite at the D17Z1 locus ofchromosome 17 (FIG. 1A) from recombinant clone p17H8 (11), isrepresentative of the general tandem molecular organization of thecomplement of α-satellites (12). The D17Z1 locus consists of an array oftandem 2.7 kb higher-order repeat units, themselves composed of 16divergent monomer sequences, each ˜171 bps in length. The 500-1000tandem repeats separated by unique EcoR1 restriction sites at theirjunctions, gives rise to arrays of 1-2 million base pairs. Less abundant15- and 14-monomer repeat units, EcoR1 heteromorphs, are also foundconstituitively on chromosome 17, with the 15- and 14-mers shown to beessentially identical in sequence to the 16-mer, excluding just 1 and 2tandem monomers, respectively (11, 13).

The chromosome 17 alpha-satellite sequence selected as the target forthird-strand binding should be unique, multicopy, andchromosome-specific. The complete nucleotide sequence of the 2.7 kbhigher-order repeat on chromosome 17 (11) shows substantial sequencedivergence (up to −30%) among its sixteen 171 bp monomers, but notwithin the 16 bp third-strand target sequence. Independent clones ofthis tandem 2.7 kb higher-order repeat are >99% identical. Moreover,sequence comparisons among the multimeric alpha-satellite higher-orderrepeats of human chromosomes show at least 15-30% divergence (12).

The purine-rich DNA target strand,

AAAAAGAAGA CAGAAG [SEQ ID NO:1] of the selected 16 bp chromosome 17α-satellite target sequence is interrupted by one pyrimidine (C)residue. Within the megabase chromosome 17 α-satellite array, this500-1000 times-repeated target sequence occurs once within monomer 16 ofeach tandem 2.7 kb higher-order repeat (11) (FIG. 1A). Within simplesequence DNA, the chance occurrence of any 16 bp sequence is 416 or 1 in4.3×10⁹. Hence, the probability of a non-alpha satellite location forthe multicopy chromosome 17 target sequence should be remote in the3×10⁹ base pair haploid human genome.

Oligodeoxyribonucleotide Third Strand Probe

The 16 nucleotide homopyrimidine third strand DNA probe:

TTTTTCTTCT TTCTTC [SEQ ID NO:2] modified at its 5′ and 3′ termini withpsoralen and biotin, respectively, binds the 16 bp α-satellite targetsequence in parallel orientation to its purine strand complement (FIG.1B). m⁵C (5-methylcytosine) residues were substituted for C to affordgreater affinity for target G•C base pairs (14). Binding specificity ofthis third strand to its purine-rich target is derived from theformation of 15 canonical triplets, 11 T:A•T and 4 m⁵C⁺:G•C. Thenon-canonical T:C•G triplet sandwiched between T:A•T nearest neighborsis only moderately destabilizing on similar Y:R•Y triplexes (4).

The [SEQ ID NO:2] third strand and its dual 5′-psoralen- and3′-biotin-modified version were synthesized by the β-cyanoethylphosphoramidite method with an Applied Biosystems Synthesizer 380B. Apsoralen (4′-hydroxymethyl-4,5′,8-trimethylpsoralen, HMT) C6phosphoramidite and biotin TEG-CPG column (both Glen Research) were usedto incorporate psoralen and biotin at respective 5′ and 3′ termini. Theannotation 5′-Pso-Tm⁵C-Bio-3′ represents dual psoralen- andbiotin-modified third strands, and 5′-Tm⁵C-3′, the non-modified form.

Crude synthesis products were analyzed by PAGE and UV shadowing.Oligomers were gel purified, desalted using Water's Sep Pak C18cartridges, lyophilized, and stored at −20° C. in sterile water. Theconcentration of oligonucleotides was determined in ddH₂O at A₂₆₀ usingextinction coefficients of 8800 and 5700 M⁻¹ cm⁻¹ for dT and dm⁵Cresidues, respectively.

Cocktail for Third-Strand In Vitro and In Situ Hybridization

Both quantitative solution and in situ third-strand hybridization assayswere carried out in 25 μl of 10 mM Bis-Tris HCl-buffered cocktail at pH6.0 containing optimal concentrations of mono-, di-, and polyvalentcations (50 mM K⁺/10 MM Mg²⁺/1 μM spermine⁴⁺), reducing and chelatingagents (1 mM DTT and 1 mM EDTA), a molecular crowding agent (2% PEG8000), deionized formamide (1.0-2.5%), and alcohol-precipitated (100-300nt) total yeast RNA (1.0 ng). For in situ hybridization, the chaotropicagent formamide was used at low concentrations (≦2.5%) to reduce thecapillary adhesion between glass microscope slide and coverslipsurfaces, so as to promote third-strand equilibration across the totalhybridizable surface area. Formamide at these concentrations lowers theTm of duplex DNA by only 0.7-1.75° C. Yeast RNA was used to compete outnon-specific binding of third strands to glass surfaces and cellulardebris. Neither deionized formamide nor yeast RNA at theseconcentrations affect the specificity or affinity of third-strandbinding to the p17H8 chromosome 17 target sequence in solution.

Third-Strand In Vitro Hybridization and Binding Specificity

Optimized solution binding assays confirmed third-strand bindingspecificity to chromosome 17 α-satellite target sites in various sizedrecombinant DNAs (5.7 to 50 kb) (15). One such assay is shown todemonstrate third-strand binding specificity to this target in asupercoiled plasmid; another, to confirm that this specificity is notdiminished in the presence of a human chromatin background similar inDNA sequence complexity to a fixed protein-depleted mitotic spread.

Thus, incubation of radiolabeled third-strand probe lacking psoralen andbiotin moieties, 5′-Tm⁵C-3′, with different supercoiled plasmidsubstrates demonstrated that third-strand binding is specific for theplasmid carrying the 16 bp α-satellite target sequence. Plasmidsubstrates were p17H8 (5.7 kb), which contains the complete 2.7 kbchromosome 17 α-satellite higher-order repeat, and pMJ1 (˜5.5 kb), ap17H8 deletion-derivative lacking the target sequence and an additional158 bp of monomer 16 but containing monomers 1-15. Supercoiled p17H8 andpMJ1 were mixed with ³²P-end-labeled third strand at an oligomer:plasmidmolar ratio of 1:1 in 1×triplex cocktail±formamide (0.25 or 2.5%) andyeast RNA (1.0 ng) at pH 6.0 and 23° C. for ˜20 hrs to attainequilibrium. FIG. 2A shows ³²P-label at gel positions corresponding tosupercoiled (sc) and open circular (oc) p17H8 (lanes 2-4). The deletionplasmid pMJ1, which lacks the target sequence but contains monomers1-15, was not bound by the labeled third strand (lanes 5-7).

The degree of sequence similarity between each of the 16-171 bp monomersthat make up the α-satellite higher-order repeat of chromosome 17provides an “internal control” for third-strand binding specificity.Sequences in monomers 1-15 are similar in length and base composition tothe monomer 16 target sequence, one differing from it by only two bases(11). Hence, this solution binding assay shows that, under theconditions employed, third-strand binding is highly specific for the 16bp sequence within monomer 16 of the 2.7 kb chromosome 17 α-satelliterepeat on p17H8.

The specificity of third-strand binding to p17H8 was also examined inthe presence of increasing amounts of unsorted human metaphasechromosomes (1-22, XY). In these solution binding assays, thestoichiometry of ³²P-end-labeled third strand, 5′-Tm⁵C-3′, to p17H8 was2:1. This permitted any non-specific binding to chromatin DNA, histoneand non-histone proteins, or RNA to be detected as decreasedplasmid-bound ³²P-third strand. The amount of alpha-satellite targetsequence due to chromosome 17 (˜1:24) was insufficient to competeagainst plasmid targets. After 1 hr incubations, plasmid signalintensity was observed to decrease slightly as unsorted chromosomesincreased from ˜24 to ˜2,400. FIG. 2B shows the results of a kineticexperiment that examined this rate-limiting effect on binding to plasmidin the presence of ˜2,400 unsorted metaphase chromosomes. It can be seenthat sc and oc plasmid-bound ³²P-third-strand signals increase overtime, t_(o)=1 hr. Thus, ˜2,400 chromosomes, i.e., 6×10¹¹ bps of unsortedhuman chromosomal DNA, did not detectably inhibit or impair thespecificity of third-strand binding to p17H8. It merely slowed thekinetics. This result suggested that specificity was achievable forthird-strand binding to unique and accessible alpha-satellite targetsequences on chromosome 17 in mitotic spreads and nuclei.

Protocol for Third-Strand In Situ Hybridization (TISH)

TISH was performed at pH 6.0 under non-denaturing solution conditionsthat give quantitative third-strand binding to recombinant DNAmolecules. A general protocol is as follows:

Solution Conditions: 1×triplex cocktail for TISH contains 10 mMBis-Tris-HCl/50 mM KCl/10 mM MgCl₂/1 mM DTT/1 mM EDTA/1 μM spermine/2%PEG 8000, pH 6.0 at 23° C. TISH pre-hybridization, post-hybridization,and UVA irradiation buffers are slight variations of this cocktail. ThepH of Bis-Tris buffers is temperature sensitive, ΔpH/Δt=−0.014 (pHunits/°C.). At the elevated temperatures used for hybridization (45° C.)and post-hybridization washing (51° C.), the pH of TISH buffers is 5.7and 5.6, respectively.

Pre-hybridization: Room temperature slides containing metaphase spreadsand interphase nuclei were dehydrated in successive 2 minute immersionsin 70, 80, and 95% ethanol (at 23° C.) incubations. After air-drying,the slides were incubated in TISH pre-hybridization buffer [10 mMBis-Tris-HCl/50 mM KCl/2.5 mM EDTA/1 mM DTT/pH 6.0] at room temperaturefor 30 minutes to promote chromosome swelling. For all remaining steps,the slides were never allowed to dry.

Hybridization: TISH hybridization mixtures (final vol. 25 μl) contain1×triplex cocktail (pH 6.0) supplemented with 1.0-2.5% deionizedformamide (BRL, ultrapure), 1.0 ng of blocking RNA (alcoholprecipitated, size fractionated total yeast RNA), and ˜20 ng ofthird-strand probe. Hybridization mixtures and slides were pre-heated at45° C. for 15 minutes in a water bath and humidified chamber,respectively, after which the mixtures were applied to the slides under22 mm² glass coverslips. Hybridization was performed in the pre-warmedhumidified chamber for 2 hours at 45° C.

Post-Hybridization Washing: Slides with coverslips intact were immersedinto TISH washing buffer [10 mM Bis-Tris-HCl/75 mM KCl/10 mM MgCl₂/1 mMDTT/pH 5.6] at 51° C., agitated to loosen the coverslips, and incubatedfurther for 5 min.

UVA Photochemistry: After removal from the wash buffer, slides werequickly covered with 22 mm² coverslips to which 25 μl ofDTT/EDTA-depleted 1×cocktail, pH 6.0, was applied. The slides were thenplaced in an open petri dish and UVA (320-400 nm) irradiated for 5minutes (14 watt low pressure mercury arc, model #RPR 3500 Å, TheSouthern New England Ultraviolet Company, Branford, Conn.). Typical UVirradiance was 1.6 J/cm² per 5 minutes at 320-400 nm. Afterwards, theslides with coverslips intact were placed in 1×PBD, pH 8.0 (phosphatebuffer with 0.05% Nonidet P40) at room temperature and agitated toloosen the coverslips. The incubation time in 1×PBD is not critical.Incubation times were generally 2-5 minutes, the longest 1 hour.

Cytochemical Detection: Slides were removed from the 1×PBD, and 60 μl offluorescein (FITC)-labeled avidin (Oncor, Inc., Gaithersburg, Md.) wasapplied under a plastic coverslip. After incubation in a pre-warmedhumidified chamber at 37° C. for 20 minutes, their plastic coverslipswere carefully removed, and the slides then washed three times for 2minutes each in 1×PBD, pH 8.0, at room temperature. After the finalwash, excess fluid was blotted from the slide edges, and 15 μl ofpropidium iodide (PI)/antifade, pH 8.5, (final PI conc. 0.3 mg/ml)(Oncor, Inc.) was applied under a 22 mm² coverslip. The slides were thenincubated for 5 minutes at room temperature.

Epi-fluorescent Microscopy: Fluorescent signals were captured and storedusing an Intel pentium powered Oncor Archive 2.0 color imaging system(Oncor, Inc.) consisting of a Zeiss Epi-fluorescent microscope equippedwith a 3CCD high resolution color camera controlled by a digital imageprocessor.

Hydrated slides containing non-denatured metaphase spreads andinterphase nuclei, isolated by standard cytogenetic methods from humanlymphocytes, and a hybrid mouse×human chr. 17 fibroblast cell line (cellline GM10498, Coriell Institute for Medical Research, Camden, N.J.),were incubated with 25 μl of 1×triplex hybridization mixtures containing20 ng of chromosome 17 α-satellite-specific third strand probe,5′-Pso-Tm⁵C-Bio-3′. Optimal parameters for TISH: pre-hybridizationhydration (30 minutes at 23° C.), hybridization (2 hours at 45° C.),post-hybridization washing (5 minutes at 51° C.), and UVA (320-400 nm)irradiation (5 minutes) were determined empirically. Post-hybridizationwashing at elevated temperature and volume effectively promotesdissociation of dual psoralen- and biotin-modified third strands boundto non-target duplex sequences. Subsequent UVA-induced psoralen-DNAphotoadduct formation prevents dissociation of target-specific boundthird strands containing m⁵C⁺ residues at the alkaline pH conditionsrequired for pre-detection wash and FITC-avidin detection. Chromosome 17centromere-specific fluorescence hybridizations with high signal tonoise ratios were obtained in the absence of RNAse A and proteasetreatment. Moreover, no signal amplification was required.

Two approaches were taken to establish that the DNA of non-denaturedfixed metaphase spreads and interphase nuclei prepared for TISH are in aduplex state. One exploits fluorescent in situ hybridization (FISH),which requires single-stranded targets. FISH assays performed onnon-denatured spreads and nuclei using a standard chromosome 17D17Z1-specific ˜200 bp biotin-labeled probe produced neithercentromere-specific nor non-specific fluorescent signals (FIG. 3). Theother is based on the general chromosome staining intercalator propidiumiodide (PI), which has higher affinity and exhibits greater fluorescencewhen bound to double-stranded (ds) than to single-stranded (ss) DNA(16). Counterstaining of TISH-assayed non-denatured spreads and nucleiwas performed at PI concentrations used for FISH, in which caserenaturation in 70% formamide is not quantitative, and should yield amixture of ds and ss DNA. Non-denatured TISH spreads emittedsignificantly brighter PI fluorescence than renatured FISH spreads.Together, these observations confirm a duplex state for both chromosome17 α-satellite and the totality of mitotic chromosomal DNA on slidesprepared for TISH.

Chromosome 17-Specific Identification by TISH

A typical in situ hybridization of 5′-Pso-Tm⁵C-Bio-3′ to a non-denaturedfixed metaphase spread and nuclei from a karyotypically normal 46, XYmale lymphocyte cell line is shown in FIGS. 4A and B. Detection did notrequire amplification. The two distinct centromere-specific yellow-greenfluorescent spots identifying homologous chromosomes 17 in the metaphasespread and three interphase nuclei were reproduced in ˜90% of allspreads and nuclei examined. The other 10% emitted short-livedfluorescent signals that were not capturable due to quenching. More than250 combined spreads and nuclei per individual examined (5 subjects intotal) were scored to ensure statistical significance. Metaphase andprometaphase (not shown) chromosomes 17 were equally labeled. Thespatial organization of the fluorescent signals in the three malesubject nuclei, one peripherally located, the other more central, wasconsistent for the bulk of nuclei examined. The spreads and nuclei inFIGS. 4A and B show the empirically determined fluorescence maximumobserved after a 2 hour hybridization at 45° C. with 20 ng of probe.Longer hybridizations with 20 ng or more probe did not elicit strongerfluorescent signals, while less time and/or probe produced weakersignals. Under TISH hybridization conditions, saturation of allaccessible chromosome 17 α-satellite targets was reproducibly achievedwithin 2 hours.

The specificity of third-strand probe binding to in situ mitoticchromosomes 17 was additionally confirmed. One approach involvedmouse×human somatic cell hybrid metaphase spreads and nuclei containinga single human chromosome 17. Another involved TISH competition assays,in which increasing concentrations of non-covalently modified thirdstrand, 5′-Tm⁵C-3′, were added to compete against5′-Pso-Tm⁵C-Bio-3′-specific binding. FIGS. 5A and B show thecentromere-specific third strand-based fluorescence of single humanmetaphase and interphase chromosome 17s in a mouse genomic background. Acompetition assay (FIG. 5C) demonstrates that when 5′-Pso-Tm⁵C-Bio-3′ iscompeted out, there is no non-specific binding to the remaining humanmitotic chromosomes in spreads.

α-Satellite Target Sequence Polymorphism Detected by TISH

Third-strand in situ hybridizations were performed on non-denaturedmetaphase spreads and nuclei of 5 unrelated individuals, 4 male and 1female. Three centromere-specific chromosome 17 fluorescent signalpatterns were recorded: (+17/+17 homozygotes) when each chromosome 17homologue was labeled, (+17/−17 heterozygotes) when only one homologuewas labeled, and (−17/−17 homozygotes) when neither emitted detectablefluorescence. In contrast, all five individuals yielded detectable+17/+17 FISH-based signals with a ˜200 bp biotin-labeled D17Z1α-satellite probe. FIGS. 6A-D show contrasting TISH and FISH images fortwo signal patterns. The two variant forms of chromosome 17 (+17 and−17) identified by TISH represent at least two distinct D17Z1haplotypes.

Inter- and intra-homologue sequence variations identified within theD17Z1 loci of chromosomes 17 can alter effective third-strand targetcopy number, and so could account for the two variant D17Z1 haplotypes.Inter-homologue sequence variations within α-satellite higher-orderrepeats of paired chromosomes 17 are characterized by single pointmutations in either simple or restriction-enzyme sequences (RFLPs), andas differences in repeat length due to deletions of single or multiplecontiguous ˜171 bp monomer units (8, 17-19). Data from Warburton andWillard (20) suggest that a partial explanation for the observedvariations in third-strand binding to different D17Z1 loci could betarget sequence heterogeneity due to a single A→G transition mutation ofthe second residue (counting 5′→3′) of the purine-rich target strand.5′-Pso-Tm⁵C-Bio-3′ third-strand binding affinity would likely besignificantly impaired by this mutation at the TISH hybridizationtemperature of 45° C. since a T:G•C mismatch replaces a canonical T:A•Ttriplet. In fact, a single T:G•C mismatch in a similar Y:R•Y nearestneighbor environment prohibited third-strand binding at ambient andelevated temperatures (4).

Intra-homologue sequence variation within the megabase ∝-satellite arrayof individual chr. 17s (18) is characterized by highly amplifiedlocalized homogeneous domains containing one distinct type ofhigher-order repeat unit differing in either sequence or length fromflanking repeat units. The existence within different chr. 17∝-satellite arrays of such domains containing the altered targetsequence (A→G transition) flanked by repeats containing wild-type targetsequences could, depending on their respective ratios, further accountfor the observed variability in third-strand binding. D17Z1 loci havingmany fewer wild-type target sequences might be expected to exhibitreduced TISH-based fluorescent signals or even none at all at 45° C. Inthis respect, TISH affords a sensitivity not inherent in FISH.

Third-Strand Target Accessibility

Third-strand probe binding to duplex targets in solution can bedescribed by the following equilibrium:${Ts} + {D\underset{k_{1{off}}}{\overset{k_{2{on}}}{\underset{\rightarrow}{\leftarrow}}}{Ts}\quad D}$

where Ts is the third strand, D is the duplex target, TsD the triplex,and k2on and k1off second-order association and first-order dissociationrate constants. When third strand concentration is in excess to a duplextarget, and binding is strong, the reaction is essentially irreversible,and like Watson-Crick reassociation kinetics, pseudo-first-order. Suchis the case for third strand titrations to a duplex target of equallength, and to single targets contained within relatively large DNAfragments (≦400 base pairs) or supercoiled plasmids (3). For reactionsinvolving such molecules, the equilibrium lies far to the right.

The TISH experiments described here demonstrate that a 16 residue thirdstrand can bind with specificity to a unique multicopy centromerictarget sequence on metaphase and interphase chr. 17s. This resultsuggests that solvent-exposed areas of non-denatured fixed mitoticchromosomes on slides are accessible to and explorable by third strands.The reproducibility of chr. 17 centromere-specific fluorescent labelingwith high signal to noise ratios confirms that third strands canassociate with specific chromosomal sequences and dissociate fromnon-specific ones on slides under solution conditions. Thisaccessibility suggests that in such spreads there are no significantbarriers to third strands directed to non-∝-satellite target sequences.

References

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2. A. G. Letai, M. A. Palladino, E. Fromm, V. Rizzo, J. R. Fresco,Biochemistry 27, 9108 (1988).

3. L. J. Maher, P. B. Dervan, B. Wold, Biochemistry 29, 8820 (1990); P.W. Roberts and D. M. Crothers, Proc. Natl. Acad. Sci. USA. 88, 9397(1991); S. F. Singleton and P. B. Dervan, Biochemistry 31, 10995 (1992);S. F. Singleton and P. B. Dervan, J Am Chem Soc, 114, 6957 (1992); M.Rougee, B. Faucon, J. L. Mergny, F. Barcelo, C. Giovannangeli, T.Garestier, C. Hélène, Biochemistry 31, 9269 (1992).

4. J. A. Fossella, Y. J. Kim, H. Shih, E. G. Richards, J. R. Fresco,Nucleic Acids Res. 21, 4511 (1993); K. Yoon, C. A. Hobbs, J. Koch, M.Sardaro, R. Kutny, A. L. Weis, Proc. Natl. Acad. Sci. USA. 89, 3840(1992); G. C. Best and P. B. Dervan, J Am Chem Soc 117, 1187 (1995).

5. E. H. Postel, S. J. Flint, D. J. Kessler, M. E,. Hogan, Proc. Natl.Acad. Sci. USA. 88, 8227 (1991); W. M. McShan, R. D. Rossen, A. H.Laughter, J. Trial, D. J. Kessler, J. G. Zendegui, M. E. Hogan, F. M.Orson, J. Biol. Chem. 267, 5712 (1992); C. Giovannangeli, S. Diviacco,V. Labrousse, S. Gryaznov, P. Charneau, C. Hélène, Proc. Natl. Acad.Sci. USA. 94, 79 (1997).

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10. The Genetics Computer Group (GCG) software from the University ofWisconsin Genetics Computer Group, Inc.

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14. T. J. Povsic and P. B. Dervan, J. Am. Chem. Soc. 111, 3059 (1989);G. E. Plum, Y. W. Park, S. F. Singleton, P. B. Dervan, K. J. Breslauer,Proc. Natl. Acad. Sci. USA. 87, 9236 (1990); L. E. Xodo, G. Manzini, F.Quadrifoglio, G. A. van der Marel, J. H. van Boom, Nucleic Acids Res.19, 5625 (1991).

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20. P. E. Warburton and H. F. Willard, Nucleic Acids Res. 20, 6033(1992).

77 1 16 DNA Artificial Sequence Description of Artificial Sequencepurine rich DNA target strand 1 aaaaagaaga cagaag 16 2 16 DNA ArtificialSequence Description of Artificial Sequence homopyrimidine third strandDNA probe 2 tttttcttct ttcttc 16 3 14 DNA Artificial SequenceDescription of Artificial Sequence Target sequences 3 agtaaaggaa agaa 144 18 DNA Artificial Sequence Description of Artificial Sequence Targetsequences 4 tatttccttt tctcgctt 18 5 15 DNA Artificial SequenceDescription of Artificial Sequence Target sequences 5 gaatgaaaag gaaag15 6 17 DNA Artificial Sequence Description of Artificial SequenceTarget sequences 6 tttccttttc tcgcttt 17 7 19 DNA Artificial SequenceDescription of Artificial Sequence Target sequences 7 aaaggtagaaaaggaaata 19 8 19 DNA Artificial Sequence Description of ArtificialSequence Target sequences 8 aaaggcagaa aaggaaata 19 9 16 DNA ArtificialSequence Description of Artificial Sequence Target sequences 9cttttctttt tcattc 16 10 20 DNA Artificial Sequence Description ofArtificial Sequence Target sequences 10 cctttctttt gagagagcag 20 11 22DNA Artificial Sequence Description of Artificial Sequence Targetsequences 11 gaaaaaggaa atatcttccc ct 22 12 17 DNA Artificial SequenceDescription of Artificial Sequence Target sequences 12 tttacccctttcttttc 17 13 14 DNA Artificial Sequence Description of ArtificialSequence Target sequences 13 gatagaaaag gaaa 14 14 14 DNA ArtificialSequence Description of Artificial Sequence Target sequences 14ggtagaaaag gaaa 14 15 23 DNA Artificial Sequence Description ofArtificial Sequence Target sequences 15 ctttccttta gaaaacagca gag 23 1614 DNA Artificial Sequence Description of Artificial Sequence Targetsequences 16 ggaaaaaaag gata 14 17 14 DNA Artificial SequenceDescription of Artificial Sequence Target sequences 17 ctttctcctt actt14 18 22 DNA Artificial Sequence Description of Artificial SequenceTarget sequences 18 gaaaagggag gtttcactct tt 22 19 21 DNA ArtificialSequence Description of Artificial Sequence Target sequences 19ctttctctac caaaagaaag g 21 20 20 DNA Artificial Sequence Description ofArtificial Sequence Target sequences 20 ggtgaaaatg gaaaaggaaa 20 21 19DNA Artificial Sequence Description of Artificial Sequence Targetsequences 21 gaggcaaatg gagaaaaag 19 22 16 DNA Artificial SequenceDescription of Artificial Sequence Target sequences 22 tcctttcttt tcattc16 23 16 DNA Artificial Sequence Description of Artificial SequenceTarget sequences 23 acagaggaaa aggaaa 16 24 16 DNA Artificial SequenceDescription of Artificial Sequence Target sequences 24 atggaggaaa aggaaa16 25 20 DNA Artificial Sequence Description of Artificial SequenceTarget sequences 25 agagatgaac ctttcttttt 20 26 18 DNA ArtificialSequence Description of Artificial Sequence Target sequences 26acggggagaa aggaaata 18 27 18 DNA Artificial Sequence Description ofArtificial Sequence Target sequences 27 atggagagaa aggaaata 18 28 19 DNAArtificial Sequence Description of Artificial Sequence Target sequences28 agagggagca gaggtgaaa 19 29 18 DNA Artificial Sequence Description ofArtificial Sequence Target sequences 29 tttctccttt ctcttcat 18 30 16 DNAArtificial Sequence Description of Artificial Sequence Target sequences30 ttccaccttt cttttc 16 31 21 DNA Artificial Sequence Description ofArtificial Sequence Target sequences 31 cctttcttga gagagagcag a 21 32 16DNA Artificial Sequence Description of Artificial Sequence Targetsequences 32 tttcaccttt ctcttc 16 33 21 DNA Artificial SequenceDescription of Artificial Sequence Target sequences 33 ccttcctttagagagagcag a 21 34 19 DNA Artificial Sequence Description of ArtificialSequence Target sequences 34 aaaggtagaa aaggaaaca 19 35 16 DNAArtificial Sequence Description of Artificial Sequence Target sequences35 gaagagaaag gtgaaa 16 36 20 DNA Artificial Sequence Description ofArtificial Sequence Target sequences 36 cctttctttt gatggaggag 20 37 17DNA Artificial Sequence Description of Artificial Sequence Targetsequences 37 ctctctctaa agaaagg 17 38 19 DNA Artificial SequenceDescription of Artificial Sequence Target sequences 38 aaaggtagaaaaggaaata 19 39 20 DNA Artificial Sequence Description of ArtificialSequence Target sequences 39 cctttctttt gatgaaggag 20 40 19 DNAArtificial Sequence Description of Artificial Sequence Target sequences40 ggaaacggga tttcttcct 19 41 20 DNA Artificial Sequence Description ofArtificial Sequence Target sequences 41 cctttctttt gatgaaggag 20 42 14DNA Artificial Sequence Description of Artificial Sequence Targetsequences 42 ggtgaaaaag ggaa 14 43 20 DNA Artificial SequenceDescription of Artificial Sequence Target sequences 43 gaaaaagggaatgtcttccc 20 44 21 DNA Artificial Sequence Description of ArtificialSequence Target sequences 44 agagtggaac ctctctcttt t 21 45 14 DNAArtificial Sequence Description of Artificial Sequence Target sequences45 ggtgagaaag gaaa 14 46 19 DNA Artificial Sequence Description ofArtificial Sequence Target sequences 46 agaggtggat ctttctttt 19 47 19DNA Artificial Sequence Description of Artificial Sequence Targetsequences 47 aaagggaata tcttcccct 19 48 14 DNA Artificial SequenceDescription of Artificial Sequence Target sequences 48 ggtgaaaagg gaaa14 49 20 DNA Artificial Sequence Description of Artificial SequenceTarget sequences 49 gaaaagggaa atatcttctc 20 50 22 DNA ArtificialSequence Description of Artificial Sequence Target sequences 50aacagagaag aaccttcctt tt 22 51 16 DNA Artificial Sequence Description ofArtificial Sequence Target sequences 51 tttcaccttt cttttc 16 52 16 DNAArtificial Sequence Description of Artificial Sequence Target sequences52 agaaagaaga cagaag 16 53 16 DNA Artificial Sequence Description ofArtificial Sequence Target sequences 53 aaaaagaaga cagaag 16 54 15 DNAArtificial Sequence Description of Artificial Sequence Target sequences54 ttttttttcc tctct 15 55 18 DNA Artificial Sequence Description ofArtificial Sequence Target sequences 55 ctttcctttc gagagaag 18 56 20 DNAArtificial Sequence Description of Artificial Sequence Target sequences56 ttttcctttc gagagagaag 20 57 21 DNA Artificial Sequence Description ofArtificial Sequence Target sequences 57 ggaaaaggaa ttatctttcc c 21 58 16DNA Artificial Sequence Description of Artificial Sequence Targetsequences 58 aaggtgaaaa aggaaa 16 59 16 DNA Artificial SequenceDescription of Artificial Sequence Target sequences 59 atggagagaa aggaaa16 60 15 DNA Artificial Sequence Description of Artificial SequenceTarget sequences 60 aaggtgaaaa agaaa 15 61 23 DNA Artificial SequenceDescription of Artificial Sequence Target sequences 61 aaaggagtagaacctttctt ttc 23 62 14 DNA Artificial Sequence Description ofArtificial Sequence Target sequences 62 ggtgaaaaag gaga 14 63 14 DNAArtificial Sequence Description of Artificial Sequence Target sequences63 ggtgaaaaag gaaa 14 64 20 DNA Artificial Sequence Description ofArtificial Sequence Target sequences 64 gaaaaaggaa atatcttccc 20 65 14DNA Artificial Sequence Description of Artificial Sequence Targetsequences 65 ggtggagaag gaaa 14 66 19 DNA Artificial SequenceDescription of Artificial Sequence Target sequences 66 ggaaaagaattatcttctc 19 67 14 DNA Artificial Sequence Description of ArtificialSequence Target sequences 67 ggtggaaaag gaaa 14 68 14 DNA ArtificialSequence Description of Artificial Sequence Target sequences 68agtggaaaag gaaa 14 69 18 DNA Artificial Sequence Description ofArtificial Sequence Target sequences 69 ttccctttca gagagcag 18 70 19 DNAArtificial Sequence Description of Artificial Sequence Target sequences70 aaaggaaata tcttcccct 19 71 22 DNA Artificial Sequence Description ofArtificial Sequence Target sequences 71 acagaaagac gagagagaag ca 22 7217 DNA Artificial Sequence Description of Artificial Sequence Targetsequences 72 ccttttcctt tatcttc 17 73 21 DNA Artificial SequenceDescription of Artificial Sequence Target sequences 73 ggaaaaggaaatatcttctc c 21 74 21 DNA Artificial Sequence Description of ArtificialSequence Target sequences 74 ggaagatggt ggaaaaggaa a 21 75 20 DNAArtificial Sequence Description of Artificial Sequence Target sequences75 ggaaaaggaa gtatcttcct 20 76 20 DNA Artificial Sequence Description ofArtificial Sequence Target sequences 76 ctataaaaag aagacagaag 20 77 20DNA Artificial Sequence Description of Artificial Sequence targetsequence 77 gatatttttc ttctgtcttc 20

What is claimed is:
 1. A method for detecting in situ the presence of atarget sequence in a substantially double-stranded nucleic acid segment,which comprises: a) contacting in situ under conditions suitable forthird strand hybridization a substantially double-stranded nucleic acidsegment with a detectable third strand, said third strand being capableof hybridizing to at least a portion of the target sequence to form atriple-stranded structure, if said target sequence is present; and b)detecting whether hybridization between the third strand and the targetduplex sequence has occurred to thereby detect the presence of thetarget sequence.
 2. The method of claim 1, wherein the third strandcomprises DNA or RNA.
 3. The method of claim 1, wherein the third strandcomprises an unnatural heterocycle base substitute, a base analog, anunnatural backbone, or a substituent which strengthens binding of thethird strand to the target sequence.
 4. The method of claim 1, whereinthe target sequence is within a human chromosome α-satellite region,other satellite regions, or non-satellite heterochromatin or euchromatinregions.
 5. The method of claim 4, wherein the target sequence is withina human chromosome α-satellite region.
 6. The method of claim 5, whereinthe target sequence is within the human chromosome 17 α-satelliteregion.
 7. The method of claim 6, wherein the third strand comprises atleast 10 contiguous bases contained within SEQ ID NO:2.
 8. The method ofclaim 6, wherein the third strand comprises the sequence of SEQ ID NO:2.9. The method of claim 1, wherein the hybridization takes place in ametaphase spread.
 10. The method of claim 1, wherein the hybridizationtakes place in an interphase nucleus.
 11. The method of claim 1, whereinthe third strand is labeled with a moiety capable of being directly orindirectly detected.
 12. The method of claim 11, wherein the thirdstrand is labeled with a compound selected from the group consisting ofbiotin, fluorescein, digoxigenin, rhodamine and phenyloxazolone.
 13. Themethod of claim 1, wherein the detection step takes place without aprior amplification step.
 14. The method of claim 1, wherein thedetection step takes place subsequent to an amplification step.
 15. Themethod of claim 1, wherein the third strand is designed to allowdetection of extra or missing chromosomes, extra or missing portions ofa chromosome, or chromosomal rearrangements.
 16. The method of claim 15,wherein the third strand is designed to allow detection of aneuploidy.17. The method of claim 16, wherein the aneuploidy is an extra ormissing human chromosome
 17. 18. The method of claim 16, wherein theaneuploidy is an extra or missing human chromosome
 21. 19. The method ofclaim 1, wherein the target sequence contains any one of SEQ ID NO:3through SEQ ID NO:75.
 20. The method of claim 1, wherein the method isdiagnostic of a genetic disorder.
 21. The method of claim 1, wherein themethod is used to screen individuals at risk for developing a disease.22. The method of claim 1, wherein the method is diagnostic of aninfectious disease.
 23. The method of claim 1, wherein the targetsequence is within a chromosomal region of a eukaryotic species or thedouble-stranded stage of a virus.
 24. The method of claim 1, wherein thethird strand comprises a moiety capable of enhancing the binding of saidthird strand to-said target sequence.
 25. The method of claim 24,wherein said moiety is a DNA binding agent, a DNA cross-linking agent,or a DNA intercalating agent.
 26. The method of claim 25, wherein themoiety is psoralen, and wherein the method further comprises the step ofirradiating the triple-stranded structure with UV light so as tocovalently affix the third strand to the target sequence.